Enzyme preparations

ABSTRACT

The invention is directed to enzyme preparations which are obtainable by providing enzyme immobilizates which comprise enzymes or microorganisms comprising enzymes immobilized on an inert support with a polyethersilicone coating obtained by hydrosilylation, to a process for preparing such enzyme preparations and to the use of enzyme preparations as an industrial biocatalyst.

CROSS REFERENCE TO RELATED APPLICATION

This application is a divisional of U.S. patent application Ser. No.12/551,975, filed Sep. 1, 2009, the entire content and disclosure ofwhich is incorporated herein by reference.

FIELD OF THE INVENTION

The invention relates to enzyme preparations which are useful asbiocatalysts.

BACKGROUND OF THE INVENTION

Microorganisms and isolated enzymes find wide use as a catalyst in thechemical industry or in food production. An overview is offered, forexample, by: A. Liese, K. Seelbach, C. Wandrey, IndustrialBiotransformations, Wiley-VCH: 2006, Weinheim, Germany.

In order to ensure economic use of such biocatalysts, some conditionshave to be satisfied: the biocatalyst has to be active for asufficiently long time under the reaction conditions, it should bereadily removable after the end of the reaction and it should bereusable as often as possible. Ideally, these conditions should besatisfied for a very wide range of reaction conditions (for exampletemperature range, type of solvents used, pressures, etc.), in order toprovide as universal as possible a catalyst.

In order to satisfy these conditions, it is typically necessary toimmobilize the enzymes or microorganisms comprising the enzymes used.

Frequently, the enzymes or microorganisms comprising the enzymes areimmobilized noncovalently on supports; the supports used are frequentlyion exchange resins or polymer particles which possess suitable particlesize distributions. Examples for this purpose are the commercialproducts Novozym 435, Lipozym RM IM or Lipozym TL IM from Novozymes A/S,Bagsvaerd, Denmark or Amano PS, from Amano, Japan. These examples areimmobilized lipases which find wide use, since such immobilizates alsoexhibit industrially utilizable activities in nonaqueous systems, i.e.,those which comprise only organic solvents, if any, as described, forexample, in J. Chem. Soc., Chem. Comm 1989, 934-935.

Patent Application DE 10 2007 031689.7 describes the variousdisadvantages of presently available immobilization technologies, inparticular and with regard to activity and stability.

SUMMARY OF THE INVENTION

The present invention discloses a novel class of enzyme preparations anda process for preparing them which overcome a large portion of thesedisadvantages. Essentially, it is stated that stable and highly activeenzyme preparations can be obtained by first adsorbing enzymes ormicroorganisms comprising enzymes on a suitable support and thenproviding this immobilizate with a silicone polymer which is accessibleby a hydrosilylation reaction.

The working examples of this application describe, however, as units forthe hydrosilylation, in addition to SiH siloxanes of various topologies,siloxanes provided only with terminally unsaturated organic radicals.

For fine adjustment of the properties of the enzyme preparations thusobtained, it would, however, be desirable also to be able to use otherunits as well as siloxane chains. A person skilled in the art is awarethat silicone compounds have particular properties, for example limitedmiscibility with other substance classes, for example many oleochemicalderivatives or water.

There is therefore still a need for methods of enzyme immobilizationwhich overcome the disadvantages of the prior art, in order to implementbiocatalytic processes which have not been achievable to date.

As such, the present invention provides enzyme preparations which do nothave one or more of the disadvantages of prior art preparations. Moreparticularly, enzyme preparations which have a comparable stability andactivity to enzyme preparations coated with pure silicone polymers, butsimultaneously also have other structural elements, are provided.

It has been found that, surprisingly, enzyme preparations that avoid oneor more drawbacks with prior art enzyme preparation can be obtained byimmobilizing enzymes or microorganisms comprising enzymes on an inertsupport and then providing them with a polyethersiloxane coatingobtained by hydrosilylating Si—H-functional polysiloxanes, the olefinicunits used being polyethers bearing at least one terminally unsaturatedgroup and, optionally additionally siloxanes which bear terminallyunsaturated organic radicals, with the proviso that at least oneolefinic unit which has at least two terminally unsaturated groups ispresent.

The present invention therefore provides enzyme preparations which areobtainable by providing enzyme immobilizates which comprise enzymes ormicroorganisms comprising enzymes immobilized on an inert support with apolyethersiloxane coating obtained by hydrosilylating Si—H-functionalpolysiloxanes using polyethers bearing at least one terminallyunsaturated group, and the use thereof as an industrial biocatalyst.

The present invention also provides a process for preparing theinventive enzyme preparations, which is characterized in that enzymeimmobilizates which comprise enzymes or microorganisms comprisingenzymes immobilized on an inert support are provided with apolyethersiloxane coating obtained by hydrosilylating Si—H-functionalpolysiloxanes using polyethers bearing at least one terminallyunsaturated group.

The inventive enzyme preparations have the advantage that they have ahigh stability with respect to mechanical forces and with respect todesorption. In spite of these improved properties, the inventive enzymepreparations have specific activities in various aqueous reactionmixtures (for example in the hydrolysis of tributyrin) and nonaqueousreaction mixtures (for example in the solvent-free synthesis of propyllaurate), which are high enough to enable industrial use.

DETAILED DESCRIPTION OF THE INVENTION

The inventive enzyme preparations and a process for preparation thereofare described below by way of example, without any intention that theinvention be restricted to these illustrative embodiments. When ranges,general formulae or compound classes are specified below, these shallnot only encompass the corresponding ranges or groups of compounds whichare mentioned explicitly but also all sub-ranges and sub-groups ofcompounds which can be obtained by selecting individual values (ranges)or compounds. When documents are cited within the present description,their contents shall be incorporated completely in thedisclosure-content of the present invention. When compounds, for exampleorganically modified polysiloxanes, which may have different units morethan once are described in the context of the present invention, theymay occur in these compounds in random distribution (statisticaloligomer) or ordered (block oligomer). Figures for the number of unitsin such compounds should be interpreted as the mean value, averaged overall appropriate compounds.

The inventive enzyme preparations are notable in that they areobtainable by providing enzyme immobilizates which comprise enzymes ormicroorganisms comprising enzymes immobilized on an inert support with asilicone coating which is obtained by hydrosilylating Si—H-functionalpolysiloxanes, the olefinic units used are polyethers bearing at leastone terminally unsaturated group and optionally additionally siloxaneswhich bear terminally unsaturated organic radicals, with the provisothat at least one olefinic unit which has at least two terminallyunsaturated groups is present.

To produce the enzyme immobilizates of the present invention, it ispossible to use whole cells, resting cells, purified enzymes or cellextracts which comprise the corresponding enzymes, or mixtures thereof.Preference is given to using hydrolytic enzymes, for example lipases,esterases or proteases, for example lipases from Candida rugosa, Candidaantarctica, Pseudomonas sp., Thermomyces lanuginosus, porcine pancreas,Mucor miehei, Alcaligenes sp., cholesterol esterase from Candida rugosa,and esterase from the porcine liver, more preferably lipases.Accordingly, the enzyme immobilizates of the invention preferablycomprise enzymes from the class of the hydrolases, preferably lipases.

The inert supports used may be inert organic or inorganic supports. Theinert supports used, or present in the enzyme immobilizate, arepreferably particulate supports which have a particle size distributionin which at least 90% of the particles have a particle size of 10 to5000 μm, preferably of 50 μm to 2000 μm. The organic supports used maybe those which comprise, or consist of, polyacrylate, polymethacrylate,polyvinylstyrene, styrene-divinylbenzene copolymers, polypropylene,polyethylene, polyethylene terephthalate, PTFE and/or other polymers.The support materials used may, depending on the enzyme to beimmobilized, be acidic or basic ion exchange resins, for example DuoliteA568, Duolite XAD 761, Duolite XAD 1180, Duolite XAD 7HP, Amberlite IR120, Amberlite IR 400, Amberlite CG 50, Amberlyst 15 (all products fromRohm and Haas) or Lewatit CNP 105 and Lewatit VP OC 1600 (products fromLanxess, Leverkusen, Germany). The inorganic supports used may be oxidicand/or ceramic supports known from the prior art. In particular, theinorganic supports used may, for example, be Celite, zeolites, silica,controlled-pore glass (CPG) or other supports, as described, forexample, in L. Cao, “Carrier-bound Immobilized Enzymes: Principles,Application and Design”, Wiley-VCH: 2005, Weinheim, Germany. Morepreferably, the inert supports present in the enzyme immobilizate or theinert supports used to produce the enzyme immobilizates consist ofsilica, polyvinylstyrene, polymethacrylate or polyacrylate.

The immobilization on the particles can, in accordance with theinvention, be effected covalently or noncovalently, preferablynoncovalently. For noncovalent immobilization, the support can beincubated or impregnated, for example, with an aqueous enzyme solutionwhich may optionally comprise further constituents, for exampleinorganic salts or detergents. This incubation/impregnation can becarried out, for example, at temperatures between 0° C. and 50° C.,preferably between 0° C. and 40° C. Preference is given to effecting theincubation/impregnation over a period of a few minutes to a few hours.The progress of the incubation can be effected by determining theconcentration of the enzyme in the solution with the common methods forprotein determination. On attainment of the desired degree ofimmobilization, the support can preferably be washed with water and, ifdesired, dried. An enzyme immobilizate obtained in this way cansubsequently be provided with a polyethersilicone coating in accordancewith the invention.

According to the invention, it is, however, also possible to use enzymeimmobilizates which are commercially available, for example Novozym 435,Lipozym RM IM or Lipozym TL IM from Novozymes A/S, Bagsvaerd, Denmark,or Amano PS from Amano, Japan.

According to the invention, the polyethersiloxane coating is obtained byhydrosilylation. To this end, preference is given to reactingSi—H-functional polysiloxanes in the presence of at least one catalyst,preferably transition metal catalysts, with polyethers which bear atleast one terminally unsaturated group.

The Si—H-functional polysiloxanes used are preferably Si—H polysiloxanesof the general formula (I)

M_(a)D_(b)D′_(c)T_(d)Q_(e)   (I)

where

-   -   M=[R¹ ₂R^(2a)SiO_(1/2)]    -   D=]R¹ ₂SiO_(2/2)]    -   D′=[R¹R^(2b)SiO_(2/2)]    -   T=[R¹SiO_(3/2)]    -   Q=[SiO_(4/2)]    -   N′=a+b+c+d+e=3 to 850, preferably 6 to 160,    -   a=2 to 10, preferably 2 to 4, especially 2,    -   b=1 to 800, preferably 2 to 150,    -   c=0 to 400, preferably 2 to 75,    -   d=0 to 10, preferably 0,    -   e=0 to 10, preferably 0,    -   R¹ are the same or different and are each independently selected        from the group comprising: saturated or unsaturated, optionally        branched alkyl groups having 1 to 30 carbon atoms, alkaryl        radicals having 7 to 30 carbon atoms, aryl radicals having 6 to        30 carbon atoms, preferably alkyl groups having 1 to 4 carbon        atoms or phenyl, especially methyl,    -   R^(2a) is independently hydrogen or R¹ and    -   R^(2b) is independently hydrogen or R¹.

The Si—H-functional polysiloxane used is preferably an Si—H polysiloxaneof the general formula (I)

where

-   -   N¹=a+b+c+d+e=6 to 160,    -   a=2,    -   b=2 to 150,    -   c=2 to 75,    -   d=0 and    -   e=0.

It is familiar to a person skilled in the art that the compounds arepresent or may be present in the form of a mixture with a distributionregulated essentially by statistical laws. The values of the indices a,b, c, d and e are therefore typically mean values.

According to the invention, the polyethers bearing at least oneterminally unsaturated group used are those of the general formula (II):

where

-   -   N²=x₁+x₂+y₁+y₂=1 to 500, preferably 2 to 200, especially 3 to        100,    -   x₁=0 to 200, preferably 1 to 200, especially 3 to 100,    -   x₂=0 to 100, preferably 1 to 100, especially 3 to 100,    -   y₁=0 to 200, preferably 1 to 200, especially 3 to 100,    -   y₂=0 to 100, preferably 1 to 200, especially 3 to 100,    -   z=0 to 500, preferably 0 to 100,    -   W=hydrogen or methyl, preferably hydrogen,    -   R⁵ is an optionally branched, optionally substituted alkylene        radical which optionally bears double bonds and has 1 to 30        carbon atoms,    -   R⁶ is independently a radical selected from the group comprising        methyl, ethyl and phenyl, preferably methyl, and    -   R⁷ is an optionally branched, optionally substituted alkyl        radical or carboxyl radical which optionally contains double        bonds and has 1 to 30 carbon atoms, with the proviso that, when        z=0, x₂=y₂=0.

It is familiar to a person skilled in the art that the compounds arepresent or may be present in the form of a mixture with a distributionregulated essentially by statistical laws. The values of the indices x₁,x₂, y₁ and y₂ are therefore typically mean values.

It is preferred that a polyether of the general formula (II) is used,the polyether consisting exclusively of ethylene glycol units, i.e.,y₁=y₂=0; equally preferably, a polyether of the general formula (II) isused, the polyether being free of ethylene glycol units, i.e., x₁=x₂=0.

It is additionally preferred that a polyether of the general formula(II) is used, where x₁+x₂ and y₁+y₂ are each greater than 1, morepreferably are each greater than 3.

It may be advantageous when polyethers of the general formula (II) inwhich z=0 are used.

In a preferred embodiment of the use of polyethers as the olefinicreactant, a polyether of the general formula (II) where z=0 is used, inwhich

-   -   R⁵ is an alkylene radical having 1 to 30 carbon atoms,        preferably having 1 to 9 carbon atoms, more preferably having 1        to 4 carbon atoms, especially methylene, and    -   R⁷ is an optionally branched, terminally unsaturated alkyl        radical having 3 to 30 carbon atoms, preferably having 3 to 11        carbon atoms, more preferably having 3 to 6 carbon atoms,        especially allyl or methallyl.

However, it may also be advantageous when polyethers of the generalformula (II) in which z>0 are used.

In a preferred embodiment of the use of polyethers as the olefinicreactant, a polyether of the general formula (II) is used, where z=1 to500, preferably 3 to 200, especially 6 to 100. In this embodiment, it ispreferred that a polyether of the general formula (II) is used, where

-   -   N²=x₁+x₂+y₁+y₂=2 to 500, preferably 4 to 200, especially 6 to        100,    -   R⁵ is an alkylene radical having 1 to 30 carbon atoms,        preferably having 1 to 9 carbon atoms, more preferably having 1        to 4 carbon atoms, especially methylene, and    -   R⁷ is an optionally branched, terminally unsaturated alkyl        radical having 3 to 30 carbon atoms, preferably having 3 to 11        carbon atoms, more preferably having 3 to 6 carbon atoms,        especially allyl or methallyl.

According to the invention, it is also possible to use mixtures of thepolyethers which bear at least one terminally unsaturated group as theolefinic reactants.

The polysiloxanes which bear terminally unsaturated organic radicalsused are preferably polysiloxanes of the general formula (III):

M_(m)D_(n)D′_(o)T_(p)Q_(q)   (III)

where

-   -   M=[R³ ₂R⁴SiO_(1/2)]    -   D=[R³ ₂SiO_(2/2)]    -   D′=[R³R⁴SiO_(2/2)]    -   T=[R³SiO_(3/2)]    -   Q=[SiO_(4/2)]    -   N²=m+n+o+p+q=3 to 1000, preferably 10 to 600,    -   m=2 to 10, preferably 2 to 4, especially 2,    -   n=1 to 800, preferably 2 to 600,    -   o=0 to 20, preferably 0 to 10, preferentially 0,    -   p=0 to 10, preferably 0,    -   q=0 to 10, preferably 0;    -   R³ are the same or different and are each independently selected        from the group comprising: saturated or unsaturated, optionally        branched alkyl groups having 1 to 30 carbon atoms, alkaryl        radicals having 7 to 30 carbon atoms, aryl radicals having 6 to        30 carbon atoms, preferably alkyl groups having 1 to 4 carbon        atoms or phenyl, especially methyl, and    -   R⁴ is independently a terminally unsaturated alkyl radical,        preferably vinyl, or an alkoxy radical, preferably having 3 to        20 carbon atoms, or R₃.

The terminal polysiloxanes which bear terminally unsaturated organicradicals used are preferably polysiloxanes of the general formula (III)where

-   -   N²=m+n+o+p+q=10 to 600,    -   M=2,    -   n=2 to 600,    -   o=0,    -   p=0,    -   q=0 and    -   R⁴ is independently a terminally unsaturated alkyl radical,        preferably vinyl.

It is familiar to a person skilled in the art that the compounds of theformula (III) are present or may be present in the form of a mixturewith a distribution regulated essentially by statistical laws. Thevalues of the indices m, n, o, p and q are therefore typically meanvalues.

When the R⁷ radical of the polyether used does not have a terminaldouble bond, compounds of the general formulae (II) and (III) are usedin a molar ratio of 0.001:1 to 1:0.001, preferably of 0.05:1 to 5:1,especially of 0.05:1 to 1:1.

When the R⁷ radical has a terminal double bond, it is also possible inaccordance with the invention to use siloxanes which bear terminallyunsaturated organic radicals. In this case, compounds of the generalformulae (II) and (III) are used in a molar ratio of 0.001:1 to 1:0,preferably of 0.05:1 to 20:1, especially of 0.10:1 to 10:1.

In the case of use of polyethers as an olefinic reactant together with afurther olefinic component of the general formula (III), preference isgiven to using a polyether of the general formula (II) where z=0, inwhich

-   -   R⁵ is an alkylene radical having 1 to 30 carbon atoms,        preferably having 1 to 9 carbon atoms, more preferably having 1        to 4 carbon atoms, especially methylene, and    -   R⁷ is an alkyl radical having 1 to 30 carbon atoms, preferably        having 1 to 9 carbon atoms, more preferably having 1 to 4 carbon        atoms, especially methyl.

The hydrosilylation can be carried out by established methods in thepresence of a catalyst. It is possible, for example, to use catalystswhich are typically used for hydrosilylations, for example platinum,rhodium, osmium, ruthenium, palladium, iridium complexes or similarcompounds or the corresponding pure elements or their derivativesimmobilized on silica, alumina or activated carbon or similar supportmaterials. Preference is given to performing the hydrosilylation in thepresence of Pt catalysts such as cisplatin or Karstedt catalyst[tris(divinyltetramethyldisiloxane)bis-platinum].

The amount of catalyst used is preferably 10⁻⁷ to 10⁻¹ mol per mole ofolefin or per mole of terminal carbon-carbon double bond, preferably 1to 100 ppm. The hydrosilylation is carried out preferably attemperatures of 0° C. to 200° C., preferably of 20° C. to 120° C.

The hydrosilylation can be carried out in the presence or absence ofsolvent. Generally, solvents are not needed for the performance of thereaction. The reaction can, however, be carried out in suitablesolvents, for example aliphatic or aromatic hydrocarbons, cyclicoligosiloxanes, alcohols or esters. Suitable solvents are especiallycyclohexane or toluene.

According to the invention, based on the mass of the support used,preferably 1 to 500% by mass, preferentially 10 to 300% by mass, morepreferably 20 to 200% by mass, of siloxane and polyether components areused. The siloxane and polyether components are composed especially ofthe sum total of the compounds of the formulae (I), (II) and (III) andof their reaction products.

The hydrosilylation can be carried out using a wide variety of differentratios of the compounds of the formula (I) to compounds of the formula(II) or to mixtures of compounds of the formulae (II) and (III).Preference is given to effecting the hydrosilylation at a molar ratiobased on the reactive groups of 1:10 to 10:1, more preferably of 1:5 to5:1, especially preferably of 1:1.5 to 1.5:1 and most preferably of 1:13to 1.3:1. Selection of the compounds of the general formulae (I), (II)and optionally (III) used and variation in their mixing ratios allowsthe properties of the polyethersilicone coating to be tailored inrelation to perviousness for substrates and other reaction properties.Selection of the weight ratio of silicone components to enzymeimmobilizates allows the layer thicknesses of the polyethersiliconecoating to be varied and to be adjusted to appropriate requirements.

The inventive polyethersiloxane coating, produced by hydrosilylation,can be obtained by carrying out the hydrosilylation in the presence ofthe enzyme immobilizates. However, it is also possible that the enzymeimmobilizates are provided subsequently with polyethersiloxanes obtainedby hydrosilylation. This can be done, for example, by treating theenzyme immobilizates with a solution of the siloxanes and of thepolyethers, for example a solution of the siloxanes and polyethers in anorganic solvent, especially cyclohexane or toluene. Subsequently, thesolvent can be removed, for example, by evaporation. The concentrationof siloxanes and polyethers in such a solution is preferably 10 to 100%by mass, more preferably 30 to 100% by mass. However, preference isgiven to obtaining the inventive polyethersiloxane coating by carryingout the hydrosilylation in the presence of the enzyme immobilizates.

The inventive enzyme preparations are preferably prepared by the processaccording to the invention described below. This process for preparingenzyme preparations is notable in that enzyme immobilizates whichcomprise enzymes or microorganisms comprising enzymes immobilized on aninert support are provided with a polyethersiloxane coating obtained byhydrosilylating Si—H-functional polysiloxanes, in which the olefinicunits used are polyethers bearing at least one terminally unsaturatedgroup and optionally additionally siloxanes which bear terminallyunsaturated organic radicals, with the proviso that at least oneolefinic unit which has at least two terminally unsaturated groups ispresent.

Preference is given to performing the process according to the inventionin such a way that the enzyme immobilizates are provided with apolyethersiloxane coating by contacting the enzyme immobilizates, underhydrosilylation conditions, with a reaction mixture which comprisesSiH-functional polysiloxanes, polyethers bearing at least one terminallyunsaturated group, and optionally polysiloxanes containing terminalcarbon-carbon double bonds, and also a catalyst which catalyzes thehydrosilylation.

Preference is given to using, in the process according to the invention,the components specified above as preferred for the inventive enzymepreparations (SiH-functional polysiloxanes, polyethers bearing at leastone terminally unsaturated group and polysiloxanes comprising terminalcarbon-carbon double bonds).

In particular, the process can be performed in such a way that ahydrosilylation reaction is carried out in the presence of enzymeimmobilizates which comprise enzymes or microorganisms comprisingenzymes immobilized on an inert support. The polyether siloxane whichforms in the hydrosilylation can provide the enzyme immobilizate with apolyethersiloxane coating.

The hydrosilylation can be carried out in a manner known to thoseskilled in the art. Preference is given to performing thehydrosilylation using the above-mentionedparameters/feedstocks/catalysts.

In a preferred embodiment of the process according to the invention, aparticular amount of enzyme immobilizate is admixed with a mixture(reaction mixture) of the silicone reagents (compounds of the formulae(I), (II) and optionally (III) plus catalyst), for example by adding amixture comprising compounds of the general formulae (I), (II) andoptionally (III), and also Karstedt catalyst. For example, a mixture ofcompounds of the formulae (I), (II) and (III) in a molar mixing ratio of1:0.6:0.6, and also Karstedt catalyst, for example 50 ppm based on theamount of silicone components present, can be added to 1 g of an enzymeimmobilizate. For the purpose of optimizing the coating properties, itmay be advantageous to dissolve the silicone components including thecatalyst before the addition in a solvent, for example cyclohexane,toluene or another organic solvent, and then to add the solution to theenzyme immobilizate. When, for example, toluene is used as the solvent,it has been found to be advantageous, after adding the solution to theenzyme immobilizates, to disperse this mixture strongly for approx. 30to 45 min, for example by means of a vortexer (Ika, level 9), until thebulk of the toluene has evaporated off. Subsequently, the resultingenzyme preparations are dried or hardened in a drying cabinet at 50° C.,for example for 12 hours. Varying the mixing ratios of the compounds ofthe general formulae (I), (II) and optionally (III) allows theproperties of the polyethersilicone coatings to be varied without anyproblem and matched to appropriate requirements.

A further embodiment of the process according to the invention differsfrom the aforementioned embodiment in that the enzyme immobilizates tobe coated are immersed into the desired reaction mixture, then removedfrom the reaction mixture and dried. The removal can be effected, forexample, using a screen which retains the enzyme immobilizate particles.The immersion time is preferably 1 to 10 minutes. The drying can beeffected in a conventional drying cabinet. Preference is given toeffecting the drying/hardening at a temperature of 20° C. to 80° C.,preferably at 40° C. to 60° C., more preferably at approx. 50° C.

In a further embodiment of the process according to the invention, whichis suitable especially for performance on the industrial scale, thehydrosilylation is carried out using a pelletizing pan unit (for examplefrom Erweka or Eirich). In this case, a defined amount of enzymeimmobilizate particles is added to the so-called pan unit and stirred.Subsequently, either the mixture comprising compounds of the formulae(I), (II) and optionally (III), and also catalyst and optionallysolvent, is added, or else, preferably, a two-substance nozzle is used(for example from Schlick or others) to apply the mixture or thecomponents under pressure (for example nitrogen or synthetic air) in theform of a fine mist of droplets, in order to ensure a very substantiallyhomogeneous distribution on the particles. After a prolonged coatingtime, the particles are removed as described above and dried or hardenedin a drying cabinet at a temperature of 20° C. to 80° C., preferably of40° C. to 60° C., more preferably of 50° C., for several hours, and canthen be stored at room temperature until further use.

In a further embodiment, the particles can be generated in a fluidizedbed reactor (for example from Glatt), in which particles and thereaction mixture are applied in appropriate mixing ratios with strongdispersion. According to the invention, this can be carried out eitherin the so-called top-down method or in the so-called bottom-up method(also known as the Wurster method).

The inventive enzyme preparations can be used, for example, asbiocatalysts, especially as industrial biocatalysts.

The examples which follow are intended to illustrate the presentinvention in detail without restricting the scope of protection which isevident from the description and the claims.

EXAMPLES

Materials and Methods:

Novozym 435 (NZ435) is a commercial enzyme immobilizate from NovozymesA/S, Bagsvaerd, Denmark, specifically a lipase B from C. antarcticaimmobilized on a polymethacrylate by adsorption.

Hydrolytic Activity (Tributyrin Hydrolysis in Aqueous Medium):

The hydrolytic activity was determined by the so-called pH-stat method.In this method, the acid released in the hydrolysis is titrated againsta base, such that the pH of the solution is kept constant. The timedependence of the consumption of base allows the acid released, andhence the enzyme activity, to be quantified. Illustrative procedure: 10to 20 mg of catalytically active particles were added to 25 ml ofTris-HCl buffer (1 mM, pH 7.5; additionally contains 0.1 mM NaCl andCaCl₂) and 500 μl of tributyrin. The hydrolytic activity was quantifiedon an autotitrator (Tritroline alpha, from Schott) via the amount ofbase titrated in (50 mM NaOH).

Synthesis Activity in PLU Units (Propyl Laurate Synthesis Activity inSolvent-Free System):

10 mg of catalytically active particles were added to 5 ml of equimolarsubstrate solution (lauric acid and 1-propanol) and incubated whileshaking and/or stiffing at 60° C. Samples (V_(sample): 50 μl) were takenevery 5 min over 25 min and transferred into 950 μl of decane (internalstandard: 4 mM dodecane). The PLUs were determined with reference to theinitial product formation rates. Propyl laurate was detected by gaschromatography (retention time: 9.791 min) (Shimadzu 2010, BTX columnfrom SGE; length 25 m, I.D. 0.22 μm; film: 0.25 μm; detector type: FIDat 300° C.; injector temperature 275° C. and injection volume 1 μl,split ratio 35.0; carrier gas pressure (helium) 150 kPa; temperatureprogramme: start temperature 60° C., hold for 1.5 min, temperature rise20° C./min, end temperature 250° C., hold for 2.5 min).

Comparative Example 1

Determination of the Desorption Stability of Conventional EnzymeImmobilizates

For the purpose of determining the desorption stability of the particlesunder harsh reaction conditions, fractions of 20.0 mg of NZ435 wereshaken (hereinafter, “incubated”) in 20 ml of MeCN/H₂O (1:1, v/v)solution at 45° C. for 30 min The particles were recovered by means of afluted filter and washed with 100 ml of H₂O, and dried at 50° C. for 12h, in order to determine the hydrolytic activity and the synthesisactivity in PLU according to the scheme described above and compare themwith the results of native NZ435 from the batch used. For arepresentation which is more clearly understandable, the results canalso be reported as a percentage with respect to the starting activity(hereinafter, “recovery rate”). The results can be taken from Table 1.

TABLE 1 Test results for comparative example 1: Synthesis Hydrolyticact. Synthesis act. Hydrolytic activity after before after incubationactivity before incubation incubation [PLU/g] incubation [U/mg] Enzymes[PLU/g] (Recovery rate) [U/mg] (Recovery rate) NZ435 4400 147 (3.3%)1.54 0.10 (6.5%) native

Example 1 Preparation of a Stable Enzyme Preparation

Illustrative Preparation:

1 g of NZ435 particles were admixed in a metal dish with the reactionmixture consisting of various compositions of compounds of the generalformulae (I), (II) and (III) (for composition see Table 2; thecomponents of the general formulae (I) and (III) were prepared byprocesses familiar to those skilled in the art, as described, forexample, in U.S. Pat. No. 7,196,153 B2, by equilibration; the monoallylpolyethers of the general formula (II) (R⁷=methyl) were prepared bymethods familiar to those skilled in the art, as described, for example,in EP 0427088 A2 or DE 2800710 A1, the diallyl polyethers of the generalformula (II) (R⁷=allyl) were purchased from Clariant, and Karstedtcatalyst (Syloff 4000, product of Dow Corning, USA). The siliconecomponents including the catalyst were each dissolved in 10 ml oftoluene before the application and then added to the particles in themetal dish. After the addition, the mixture was immediately dispersedstrongly by means of a vortexer (Ika, level 9) for 30 to 45 min, untilthe bulk of the toluene had evaporated off. Subsequently, the particleswere dried at 50° C. in a drying cabinet for about 12 h. The results areshown in Table 2. The activities of the coated particles are both basedon the unit of weight of coated particle and, for better comparability,based on the amount of native NZ435 used. Finally, the latter values areadditionally shown in a percentage ratio relative to the activity of thenative NZ435 (hereinafter “activity yield”).

TABLE 2 Composition of the different coated particles ComponentHydrolysis of the act. Synthesis general Component [U/mg of act. [PLU/gformula (I); of the general immob. of immob. Starting c = d = 0;Component of the general formula (III); (U/mg of (PLU/g of weight R¹ =R^(2a) = formula (II); b = c = d = 0; Proportion NZ435; NZ435; ofmethyl, W = H; R⁵ = —CH₂—; R⁶ = R³ = methyl, of NZ435 activity activityNo. NZ435 R^(2b) = H methyl; R⁴ = vinyl [%] yield)] yield)] NZ435¹ 1 g —— — 100 1.54 4400 (4400) i 1 g a = 43, b = 5 x₁ = 25, x₂ = y₁ = y₂ = z =0, a = 98 50 0.50 1803 490 mg R⁷ = allyl; 71.4 mgL 438.6 mg (1.00; 65%)(3606; 82%) ii 1 g a = 43, b = 5 x₁ = 25, x₂ = y₁ = y₂ = z = 0, a = 9840 0.48 1504 735 mg R⁷ = allyl; 107.1 mg 657.9 mg (1.20; 78%) (3759;85%) iii 1 g a = 43, b = 5 x₁ = 25, x₂ = y₁ = y₂ = z = 0, a = 98 50 0.471767 514.1 mg R⁷ = allyl; 38.9 mg 447 mg (0.94; 55%) (3534; 80%) iv 1 ga = 43, b = 5 x₁ = 25, x₂ = y₁ = y₂ = z = 0, a = 98 40 0.39 1592 771.3mg R⁷ = allyl; 58.4 mg 670.2 mg (0.98; 64%) (3980; 90%) v 2 g a = 43, b= 5 x₁ = 23, y₁ = 5, x₂ = y₂ = z = a = 98 40 0.47 1540 450 mg 0, 2360 mg(1.20; 78%) (3850; 88%) R⁷ = methyl; 190 mg vi 2 g a = 43, b = 5 x₁ =23, y₁ = 5, x₂ = y₂ = z = a = 98 40 0.47 1875 470 mg 0, 2430 mg (1.20;78%)  (4687; 106%) R⁷ = methyl; 100 mg vii 2 g a = 43, b = 5 x₁ = x₂ =2, y₁ = y₂ = 8, z = a = 98 40 0.41 1771 420 mg 15, R⁷ = allyl; 340 mg2240 mg (1.03; 67%)  (4428; 100%) viii 2 g a = 43, b = 5 x₁ = x₂ = 2, y₁= y₂ = 8, z = a = 98 40 0.36 1710 410 mg 15, R⁷ = allyl; 390 mg 2200 mg(0.90; 58%) (4275; 97%) ¹Data for native NZ435 taken from ComparativeExample 1

The particles prepared by this process have, compared to the untreatedimmobilizate, activity yields in the hydrolysis of 55% up to 78%(Example 1 i, 1.20 U/mg of NZ435 compared to 1.54 U/mg for untreatedNZ435, as described in Comparative Example 1). In the synthesis, verygood activity yields of 80% up to 106% were achievable.

Example 2

Determination of the Desorption Stability of Stable Enzyme Preparations

Analogously to Comparative Example 1, the particles obtained fromExample 1 were treated with water/acetonitrile and then the hydrolysisactivity and the synthesis activity based on the proportion by weight ofNZ435 in the preparation were determined The result of thisdetermination can be taken from Table 3; analogously to ComparativeExample 1, the recovery rate was also determined.

TABLE 3 Results from Example 2 Hydrolysis Synthesis act. after Synthesisact. after Pro- Hydrolysis incubation act. incubation portion act.before [U/mg of before [U/mg of of incubation NZ435]; incubation NZ435];NZ435 [U/mg of (recovery [U/mg of (recovery [%] NZ435] rate) NZ435]rate) NZ435¹ 100 1.54  0.10; (6.5%) 3761  147; (3.9%) i 50 1.00 0.38;(38%) 3606  340; (9.4%) ii 40 1.20 0.81; (68%) 3759 2402; (64%)  iii 500.94 0.28; (30%) 3534  240; (6.8%) iv 40 0.98 0.58; (59%) 3980 1500;(33%)  v 40 1.20 0.38; (32%) 3850 425; (11%) vi 40 1.20 0.43; (36%) 4587875; (19%) vii 40 1.03 0.50; (51%) 4428 603; (14%) viii 40 0.90 0.55;(61%) 4275 695; (16%) ¹Data for native NZ435 taken from ComparativeExample 1

Whereas untreated native enzyme immobilizate after incubation exhibitsalmost no hydrolysis or synthesis activity whatsoever, thepolyethersiloxane-coated particles exhibit hydrolysis activities of upto 68% of the starting activity and synthesis activities of up to 64% ofthe starting activity.

While the present invention has been particularly shown and describedwith respect to preferred embodiments thereof, it will be understood bythose skilled in the art that the foregoing and other changes in formsand details may be made without departing from the spirit and scope ofthe present invention. It is therefore intended that the presentinvention not be limited to the exact forms and details described andillustrated, but fall within the scope of the appended claims.

What is claimed is:
 1. An enzyme preparation comprising: an enzymeimmobilizate including microorganisms comprising enzymes immobilized onan inert support; and a polyethersiloxane coating located on said enzymeimmobilizate, said polyethersiloxane coating is obtained byhydrosilylating Si—H-functional polysiloxanes, in which the olefinicunits used are polyethers bearing at least one terminally unsaturatedgroup, with the proviso that at least one of the polyether groups has atleast two terminally unsaturated groups.
 2. The enzyme preparationaccording to claim 1, wherein said enzymes are hydrolases.
 3. The enzymepreparation according to claim 1, wherein the inert support has aparticle size distribution in which 90% of the particles have a particlesize of 10 to 5000 μm.
 4. The enzyme preparation according to claim 1,wherein the inert support is silica, polyvinylstyrene, polymethacrylateor polyacrylate.
 5. The enzyme preparation according to claim 1, whereinthe Si—H-functional polysiloxanes are Si—H polysiloxanes of formula (I)M_(a)D_(b)D′_(c)T_(d)Q_(c)   (I) where M=[R¹ ₂R^(2a)SiO_(1/2)] D=[R¹₂SiO_(2/2)] D′=[R¹R^(2b)SiO_(2/2)] T=[R¹SiO_(3/2)] Q=[SiO_(4/2)]a+b+c+d+e=3 to 850, a=2 to 10, b=1 to 800, c=0 to 400, d=0 to 10, e=0 to10, R¹ are the same or different and are each independently selectedfrom saturated or unsaturated, optionally branched alkyl groups having 1to 30 carbon atoms, alkaryl radicals having 7 to 30 carbon atoms, oraryl radicals having 6 to 30 carbon atoms, R^(2a) is independentlyhydrogen or R¹ and R^(2b) is independently hydrogen or R¹.
 6. The enzymepreparations according to claim 5, wherein a+b+c+d+e=6 to 160, a=2, b=2to 150, c=2 to 75, d=0 and e=0.
 7. The enzyme preparation according toclaim 1, wherein the polyethers bearing at least one terminallyunsaturated group are those of formula (II):

where x₁+x₂+y₁+y₂=1 to 500, x₁=0 to 200, x₂=0 to 100, y₁=0 to 200, y₂=0to 100, z=0 to 500, W=hydrogen or methyl, R⁵ is an optionally branched,optionally substituted alkylene radical which optionally bears doublebonds and has 1 to 30 carbon atoms, R⁶ is independently a radicalselected from methyl, ethyl or phenyl, and R⁷ is an optionally branched,optionally substituted alkyl radical or carboxyl radical whichoptionally contains double bonds and has 1 to 30 carbon atoms, with theproviso that, when z=0, x₂=y₂=0.
 8. The enzyme preparation according toclaim 1, wherein said olefinic units used further include polysiloxaneswhich bear terminally unsaturated organic radicals, and wherein thepolysiloxanes which bear terminally unsaturated organic radicals are offormula (III)M_(m)D_(n)D′_(o)T_(p)Q_(q)   (III) where M=[R³ ₂R⁴SiO_(1/2)] D=[R³₂SiO_(2/2)] D′=[R³R⁴SiO_(2/2)] T=[R³SiO_(3/2)] Q=[SiO_(4/2)] m+n+o+p+q=3to 1000, m=2 to 10, n=1 to 800, o=0 to 20, p=0 to 10, q=0 to 10, R³ arethe same or different and are each independently selected from saturatedor unsaturated, optionally branched alkyl groups having 1 to 30 carbonatoms, alkaryl radicals having 7 to 30 carbon atoms, or aryl radicalshaving 6 to 30 carbon atoms, and R⁴ is independently a terminallyunsaturated alkyl radical, preferably vinyl, or an alkoxy radical or R₃.9. The enzyme preparation according to claim 8, wherein m+n+o+p+q=10 to600, m=2, n=2 to 600, o=0, p=0, q=0, and R⁴ is independently aterminally unsaturated alkyl radical.
 10. An enzyme preparationcomprising: an enzyme immobilizate including microorganisms comprisingenzymes immobilized on an inert support; and a polyethersiloxane coatinglocated on said enzyme immobilizate, said polyethersiloxane coating isobtained by hydrosilylating Si—H-functional polysiloxanes, in which theolefinic units used are polyethers bearing at least one terminallyunsaturated group and siloxanes which bear unsaturated organic radicals,with the proviso that at least one olefinic unit of at least one of saidpolyethers and/or said siloxanes has at least two terminally unsaturatedgroups.
 11. The enzyme preparation according to claim 10, wherein theSi—H-functional polysiloxanes are Si—H polysiloxanes of formula (I)M_(a)D_(b)D′_(c)T_(d)Q_(e)   (I) where M=[R¹ ₂R^(2a)SiO_(1/2)] D=[R¹₂SiO_(2/2)] D′=[R¹R^(2b)SiO_(2/2)] T=[R¹SiO_(3/2)] Q=[SiO_(4/2)]a+b+c+d+e=3 to 850, a=2 to 10, b=1 to 800, c=0 to 400, d=0 to 10, e=0 to10, R¹ are the same or different and are each independently selectedfrom saturated or unsaturated, optionally branched alkyl groups having 1to 30 carbon atoms, alkaryl radicals having 7 to 30 carbon atoms, oraryl radicals having 6 to 30 carbon atoms, R^(2a) is independentlyhydrogen or R¹ and R^(2b) is independently hydrogen or R¹.
 12. Theenzyme preparations according to claim 11, wherein a+b+c+d+e=6 to 160,a=2, b=2 to 150, c=2 to 75, d=0 and e=0.
 13. The enzyme preparationaccording to claim 10, wherein the polyethers bearing at least oneterminally unsaturated group are those of formula (II):

where x₁+x₂+y₁+y₂=1 to 500, x₁=0 to 200, x₂=0 to 100, y₁ =0 to 200, y₂=0to 100, z=0 to 500, W=hydrogen or methyl, R⁵ is an optionally branched,optionally substituted alkylene radical which optionally bears doublebonds and has 1 to 30 carbon atoms, R⁶ is independently a radicalselected from methyl, ethyl or phenyl, and R⁷ is an optionally branched,optionally substituted alkyl radical or carboxyl radical whichoptionally contains double bonds and has 1 to 30 carbon atoms, with theproviso that, when z=0, x₂=y₂=0.
 14. The enzyme preparation according toclaim 10, wherein said siloxanes which bear unsaturated organicradicals, and wherein the polysiloxanes which bear terminallyunsaturated organic radicals are of formula (III)M_(m)D_(n)D′_(o)T_(p)Q_(q)   (III) where M=[R³ ₂R⁴SiO_(1/2)] D=[R³₂SiO_(2/2)] D′=[R³R⁴SiO_(2/2)] T=[R³SiO_(3/2)] Q=[SiO_(4/2)]N²=m+n+o+p+q=3 to 1000, m=2 to 10, n=1 to 800, o=0 to 20, p=0 to 10, q=0to 10, R³ are the same or different and are each independently selectedfrom saturated or unsaturated, optionally branched alkyl groups having 1to 30 carbon atoms, alkaryl radicals having 7 to 30 carbon atoms, oraryl radicals having 6 to 30 carbon atoms, and R⁴ is independently aterminally unsaturated alkyl radical, preferably vinyl, or an alkoxyradical or R₃.
 15. The enzyme preparation according to claim 14, whereinm+n+o+p+q=10 to 600, m=2, n=2 to 600, o=0, p=0, q=0 and R⁴ isindependently a terminally unsaturated alkyl radical.
 16. A process forpreparing an enzyme preparation, comprising coating an enzymeimmobilizate which comprises enzymes or microorganisms comprisingenzymes immobilized on an inert support with a polyethersiloxanecoating, said polyethersiloxane coating is obtained by hydrosilylatingSi—H-functional polysiloxanes, in which the olefinic units used arepolyethers bearing at least one terminally unsaturated group andoptionally additionally siloxanes which bear terminally unsaturatedorganic radicals, with the proviso that at least one olefinic unit whichhas at least two terminally unsaturated groups is present.
 17. Theprocess according to claim 16, wherein the enzyme immobilizates areprovided with a polyethersilicone coating by contacting the enzymeimmobilizates, under hydrosilylation conditions, with a reaction mixturewhich comprises Si—H-functional polysiloxanes, polyethers bearing atleast one terminally unsaturated group, optionally polysiloxanescontaining terminal carbon-carbon double bonds, and also a catalystwhich catalyses the hydrosilylation.